Acute myeloid leukemia microenvironment impairs neutrophil maturation and function through NF?B signaling [scRNA-seq]

Acute myeloid leukemia (AML), an aggressive hematological malignancy, is driven by oncogenic mutations in stem and progenitor cells that give rise to AML blasts. While these mutations are well-characterized, their impact on healthy hematopoiesis—those blood cells exposed to AML but not mutated—has not been well-characterized. As the marrow is the major site for granulopoiesis, neutrophils are heavily influenced by AML pathobiology. Indeed, most AML patients report neutropenia, rendering them susceptible to infections. However, since AML studies use peripheral blood mononuclear cells devoid of neutrophils, the characterization of neutrophil dysfunction remains poorly understood. To investigate AML-exposed neutrophils, a pre-clinical AML mouse model was used where primary leukemic cells were transplanted into non-irradiated neutrophil reporter (Ly6G-tdTomato; Catchup) hosts. Neutrophils could not completely mature, suggesting impaired granulopoiesis. Single-cell transcriptomics of AML-exposed neutrophils revealed higher inflammation signatures and expression of CD14, an inflammatory marker. To address the factors contributing to this biology, an ex vivo cytokine screen was performed on marrow neutrophils and identified that NF?B signaling drove CD14 expression. AML-exposed neutrophils displayed widespread chromatin remodeling, and de novo motif discovery predicted increased binding sites for CCAAT-enhancer-binding proteins (C/EBPs) and Interferon regulatory factors (IRFs). Moreover, AML-exposed neutrophils inhibited T-cell proliferation, highlighting their immune-suppressive capability. Finally, similar biology of immature, inflammatory neutrophils was found in AML patients, again indicating dysregulated granulopoiesis. Collectively, these data show that AML-associated inflammation alters neutrophil granulopoiesis, impairs neutrophil function, and drives immunosuppression, thus contributing to patient susceptibility to infection. Overall design: CatchUp mice were transplanted with Meis1-HoxA9 leukemic cells or CD45.2+ control bone marrow cells. Single-cell RNA-sequencing was performed 14 days after, when animals developed acute myeloid leukemia. Bone marrow neutrophils from (4) control and (4) AML mice were isolated by fluorescence-activated cell sorting (FACS) and analyzed by scRNA-seq using 10x genomics platform. In parallel, we sorted bone marrow neutrophils from healthy CatchUp mice and treated them with either GCSF, GCSF + GMCSF, or GCSF + GMCSF + IKK16 for 24 hours. We subsequencly sorted neutrophils from each condition (n=2/condition) and analyzed by scRNA-seq using 10x genomics platform.